Disclosed herein are substituted or unsubstituted 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 or 4-oximes or pharmaceutically acceptable salts thereof that have high binding to the glycine receptor and are usefully administered to animals for the treatment or prevention of the adverse consequences of the hyperactivity of the excitatory amino acids, or neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia and surgery, for the treatment of anxiety, chronic pain, convulsions, and neurodegenerative diseases including Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome; and for the inducement of anesthesia.

Claim:

What is claimed is:

1. A compound having the Formula: ##STR13## wherein one of X and Y is oxygen and the other of X and Y is N--OR, where R is

The present invention is in the field of medicinal chemistry. In particular, the present invention relates to substituted and unsubstituted 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 or 4-oximes and pharmaceutically acceptable salts thereof andtheir use to treat or prevent neuronal degeneration associated with ischemia, pathophysiologic conditions associated with neuronal degeneration, convulsions, anxiety, and chronic pain as well as to induce anesthesia and treat or prevent psychosis.

BACKGROUND OF THE INVENTION

Glutamate is thought to be the major excitatory neurotransmitter in the brain. There are three major subtypes of glutamate receptors in the CNS. These are commonly referred to as kainate, AMPA and N-methyl-D-aspartate (NMDA) receptors (Watkinsand Olverman, Trends in Neurosci. 7:265-272 (1987)). NMDA receptors are found in the membranes of virtually every neuron in the brain. NMDA receptors are ligand-gated cation channels that allow Na.sup.+, K.sup.+ and Ca.sup.++ to permeate when they areactivated by glutamate or aspartate (non-selective, endogenous agonists) or by NMDA (a selective, synthetic agonist) (Wong and Kemp, Ann. Rev. Pharmacol. Toxicol. 31:401-425 (1991)).

Glutamate alone cannot activate the NMDA receptor. In order to become activated by glutamate, the NMDA receptor channel must first bind glycine at a specific, high affinity glycine binding site which is separate from the glutamate/NMDA bindingsite on the receptor protein (Johnson and Ascher, Nature 325:329-331 (1987)). Glycine is therefore an obligatory coagonist at the NMDA receptor/channel complex (Kemp, J. A., et al., Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)).

Besides the binding sites for glutamate/NMDA and glycine, the NMDA receptor carries a number of other functionally important binding sites. These include binding sites for Mg.sup.++, Zn.sup.++, polyamines, arachidonic acid and phencyelidine(PCP) (Reynolds and Miller, Adv. in Pharmacol. 21:101-126 (1990); Miller, B., et al., Nature 355:722-725 (1992)). The PCP binding site--now commonly referred to as the PCP receptor--is located inside the pore of the ionophore of the NMDAreceptor/channel complex (Wong, E. H. F., et al., Proc. Natl. Acad. Sci. USA 83:7104-7108 (1986); Huettner and Bean, Proc. Natl. Acad. Sci. USA 85:1307-1311 (1988); MacDonald, J. F., et al., Neurophysiol. 58:251-266 (1987)). In order for PCP togain access to the PCP receptor, the channel must first be opened by glutamate and glycine. In the absence of glutamate and glycine, PCP cannot bind to the PCP receptor although some studies have suggested that a small amount of PCP binding can occureven in the absence of glutamate and glycine (Sircar and Zukin, Brain Res. 556:280-284 (1991)). Once PCP binds to the PCP receptor, it blocks ion flux through the open channel. Therefore, PCP is an open channel blocker and a non-competitive glutamateantagonist at the NMDA receptor/channel complex.

One of the most potent and selective drugs that bind to the PCP receptor is the anticonvulsant drug MK-801. This drug has a K.sub.d of approximately 3 nM at the PCP receptor (Wong, E. H. F., et al., Proc. Natl. Acad. Sci. USA 83:7104-7108(1986)).

However, the therapeutic potential of these PCP receptor drugs as ischemia rescue agents in stroke has been severely hampered by the fact that these drugs have strong PCP-like behavioral side effects (psychotomimetic behavioral effects) whichappear to be due to the interaction of these drugs with the PCP receptor (Tricklebank, M. D., et al., Eur. J. Pharmacol. 167:127-135 (1989); Koek, W., et al., J. Pharmacol. Exp. Ther. 245:969 (1989); Willets and Balster, Neuropharmacology 27:1249(1988)). These PCP-like behavioral side effects appear to have caused the withdrawal of MK801 from clinical development as an ischemia rescue agent. Furthermore, these PCP receptor ligands appear to have considerable abuse potential as demonstrated bythe abuse liability of PCP itself.

An alternate way of inhibiting NMDA receptor channel activation is by using antagonists at the glycine binding site of the NMDA receptor. Since glycine must bind to the glycine site in order for glutamate to effect channel opening (Johnson andAscher, Nature 325:329-331 (1987); Kemp, J. A., et al., Proc. Natl. Acad. Sci. USA 85:6547-6550 (1988)), a glycine antagonist can completely prevent ion flux through the NMDA receptor channel--even in the presence of a large amount of glutamate.

Recent in vivo microdialysis studies have demonstrated that in the rat focal ischemia model, there is a large increase in glutamate release in the ischemic brain region with no significant increase in glycine release (Globus, M. Y. T., et al., J.Neurochem. 57:470-478 (1991)). Thus, theoretically, glycine antagonists should be very powerful neuroprotective agents, because they can prevent the opening of NMDA channels by glutamate non-competitively and therefore--unlike competitive NMDAantagonists--do not have to overcome the large concentrations of endogenous glutamate that are released in the ischemic brain region.

B. The only widely available glycine antagonist that sufficiently penetrates the blood/brain barrier--the drug HA-966 (Fletcher and Lodge,. Eur. J. Pharmacol. 151:161-162 (1988))--is a partial agonist with micromolar affinity for the glycinebinding site. A neuroprotective efficacy for HA-966 in vivo has not been demonstrated nor has it been demonstrated for the other available glycine antagonists because they lack bioavailability in vivo.

A need continues to exist for potent and selective glycine/NMDA antagonists which can penetrate the blood/brain barrier and which:

lack the PCP-like behavioral side effects common to the PCP-like NMDA channel blockers such as MK801 or to the competitive NMDA receptor antagonists such as CGS19755;

show potent anti-ischemic efficacy because of the non-competitive nature of their glutamate antagonism at the NMDA receptor;

have utility as novel anticonvulsants with fewer side-effects than the PCP-like NMDA channel blockers or the competitive NMDA antagonists;

help in defining the functional significance of the glycine binding site of the NMDA receptor in vivo.

There have been a number of reports in the literature regarding the preparation of 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 and 4-oximes. For example, Fadda, A. A. et al, Pharmazie 46:743-4 (1991), disclose compounds having the formula:##STR1## These compounds were employed as intermediates for the preparation of benzenesulphonyl derivatives with antibacterial and anticandidal activities. See also, Fadda, A. A. et al., J. Indian Chem. Soc. 68:393-5 (1991).

Hardman and Partridge, J. Chem. Soc. 614-20 (1958), disclose 7-chloro-1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oxime. Hardman and Partridge report that most of the quinoline derivatives described in their communication were examined foramoebacidal activity, but that no activity was observed.

Stankevicius, A. et al., Chem. Abstr. 115:114174h (1991), disclose compounds having the formula: ##STR2## wherein R' is arylsulfonyl and A may be --NHCO-- (1,2,3,4-tetrahydroquinoline-2,3,4-trione-4-oxime). These compounds are reported to beuseful as intermediates for the preparation of cyanocarboxylic acid amides.

SUMMARY OF THE INVENTION

The invention relates to a method of treating or preventing neuronal loss associated with stroke, ischemia, CNS trauma, hypoglycemia and surgery, as well as treating neurodegenerative diseases including Alzheimer's disease, amyotrophic lateralsclerosis, Huntington's disease and Down's syndrome, treating or preventing the adverse consequences of the overstimulation of the excitatory amino acids, as well as treating anxiety, convulsions, chronic pain; psychosis and inducing anesthesia,comprising administering to an animal in need of such treatment a compound of the Formula (I) ##STR3## or a pharmaceutically acceptable salt thereof;

wherein one of X or Y is oxygen and the other of X or Y is N--OR, where R may be hydrogen, alkyl, aryl, heteroaryl, acyl, halogen-substituted acyl or aryloyl; and

The present invention resulted from the initial discovery that 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oxime and 1,2,3,4-tetrahydroquinoline-2,3,4-trione-4-oxime exhibit high binding to the glycine receptor. Since1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 and 4-oxime are highly selective for the glycine receptor, it is expected that the compounds of the present invention may not exhibit the PCP-like behavioral side effects common to the PCP-like NMDA channelblockers such as MK-801 and other NMDA antagonists such as CGS19755. Thus, the compounds of the present invention are useful for treating, pathophysiologic conditions, without significant side effects or toxicity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to substituted and unsubstituted 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 or 4-oximes which are highly selective competitive antagonists of the glycine binding site of the NMDA receptor and of the excitatory aminoacids. The 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oximes which may be employed in the practice of the invention have the following Formula (II): ##STR4## or a pharmaceutically acceptable salt thereof;

Of course, it is to be understood that R.sub.1 -R.sub.4 may be the same or different. It is also to be understood that the oximes of the present invention exist as syn and/or anti isomers. The present invention is also directed to such isomersand mixtures of such isomers.

The 1,2,3,4-tetrahydroquinoline-2,3,4-trione-4-oximes which may be used in the practice of the present invention have the following Formula (II): ##STR5## wherein R and R.sub.1 -R.sub.4 are as defined above.

Preferred compounds within the scope of Formulae II and III are wherein R.sub.1 and R.sub.3 are other than hydrogen, e.g., R.sub.1 is nitro, amino, chloro or bromo; R.sub.2 is hydrogen, chloro, bromo or trifluoromethyl; R.sub.3 is nitro, chloro,bromo or trifluoromethyl; and R.sub.4 is hydrogen, nitro, chloro, bromo or amino.

Typical aryloxy groups include any of the C.sub.6-14 aryl groups linked by oxygen to any of the 5-, 6-, 7- and/or 8- positions of the quinoline ring, e.g. phenoxy and 1-naphthyloxy groups.

Typical substituted aryl groups include any of the C.sub.6-14 aryl groups substituted by one or more halo, nitro, cyano, alkyl, alkenyl, and alkynyl groups, e.g. 2-chlorophenyl, 2,4-dibromophenyl and the like.

Typical substituted aryloxy groups include any of the C.sub.6-14 aryl groups substituted by one or more halo, nitro, cyano, alkyl, alkenyl, and alkynyl groups, and linked by oxygen to any of the 5-, 6-, 7- and/or 8- positions of the quinolinering, e.g. 2-chlorophenoxy, 2,4-dibromophenoxy and the like.

Typical aryloyl groups include any of the above-mentioned aryl groups substituted by a carbonyl group.

Typical heteroaryloxy groups include any of the heteroaryl groups linked by oxygen to any of the 5-, 6-, 7- and/or 8- positions of the quinoline ring, e.g. 2-furanoxy, 4-pyridoxy, 2-pyrazinoxy, purine-6-oxy and the like.

Typical alkoxy groups include any one of the C.sub.1-4 alkyl groups mentioned above linked by oxygen to any of the 5-, 6-, 7- and/or 8- positions of the quinoline ring.

Typical haloalkoxy groups include any one of the alkoxy groups substituted by one or more fluoro, chloro, bromo or iodo groups, e.g., trifluoromethoxy, trichloromethoxy, 2-chloroethoxy, 2-bromoethoxy, pentafluoroethyl, 3,3,3-trichloropropoxy,4,4,4-trichlorobutoxy and the like.

Typical trialkylsilyl-substituted alkoxy groups include any one of the C.sub.1-4 alkoxy groups substituted by a C.sub.3-6 trialkylsilyl group, e.g. 2-trimethylsilylethoxy, 2-triethylsilylethoxy and 2-(t-butyldimethlsilyl)ethoxy and the like.

Certain of the compounds of the present invention are expected to be potent anticonvulsants in animal models and will prevent ischemia-induced nerve cell death in the gerbil global ischemia model after i.p. administration.

The compounds of the present invention are active in treating or preventing neuronal loss, neurodegenerative diseases, chronic pain, are active as anticonvulsants and antipsychotics and for inducing anesthesia. Certain of the compounds of thepresent invention are expected to exhibit little or no untoward side effects caused by non-selective binding with other receptors, particularly, the PCP and glutamate receptors associated with the NMDA receptor. In addition, certain of the compounds mayblock kainate, AMPA and quisqualate receptors and are therefore useful as broad-spectrum excitatory amino acid receptor antagonists. Moreover, the compounds of the present invention are effective in treating or preventing the adverse consequences of thehyperactivity of the excitatory amino acids, e.g., those which are involved in the NMDA receptor system, by blocking the glycine receptors and preventing the ligand-gated cation channels from opening and allowing excessive influx of Ca.sup.++ intoneurons, as occurs during ischemia.

Neurodegenerative diseases which may be treated with the compounds of the present invention include those selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Down's syndrome.

The compounds of the present invention find particular utility in the treatment or prevention of neuronal loss associated with multiple strokes which give rise to dementia. After a patient has been diagnosed as suffering from a stroke, thecompounds of the present invention may be administered to ameliorate the immediate ischemia and prevent further neuronal damage that may occur from recurrent strokes.

Moreover, the compounds of the present invention are able to cross the blood/brain barrier which makes them particularly useful for treating or preventing conditions involving the central nervous system.

The compounds of the invention find particular utility in treating or preventing the adverse neurological consequences of surgery. For example, coronary bypass surgery requires the use of heart-lung machines which tend to introduce air bubblesinto the circulatory system which may lodge in the brain. The presence of such air bubbles robs neuronal tissue of oxygen, resulting in anoxia and ischemia. Pre- or post- surgical administration of the 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oximesof the present invention will treat or prevent the resulting ischemia. In a preferred embodiment, the compounds of the invention are administered to patients undergoing cardiopulmonary bypass surgery or carotid endarterectomy surgery.

The compounds of the present invention also find utility in treating or preventing chronic pain. Such chronic pain may be the result of surgery, trauma, headache, arthritis, or other degenerative disease. The compounds of the present inventionalso find particular utility in the treatment of phantom pain that results from amputation of an extremity. In addition to treatment of pain, the compounds of the invention are also expected to be useful in inducing anesthesia, either general or localanesthesia, for example, during surgery.

The glycine and excitatory amino acid antagonists may be tested for in vivo anticonvulsant activity after intrapedtoneal injection using a number of anticonvulsant tests in mice (audiogenic seizure model in DBA-2 mice, pentylenetetrazol-inducedseizures in mice, NMDA-induced death). The compounds may also be tested in drug discrimination tests in rats trained to discriminate PCP from saline. It is expected that most of the compounds of the present invention will not generalize to PCP at anydose. In addition, it is also expected that none of the compounds will produce a behavioral excitation in locomotor activity tests in the mouse. It is expected that such results will suggest that the glycine, AMPA, kainate and quisqualate antagonistsof the present invention do not show the PCP-like behavioral side effects that are common to NMDA channel blockers such as MK-801 and PCP or to competitive NMDA antagonists such as CGS19755.

The glycine and excitatory amino acid antagonists are also expected to show potent activity in vivo after intraperitoneal injection suggesting that these compounds can penetrate the blood/brain barrier.

The compounds of the present invention may be tested for potential glycine antagonist activity by observing the inhibition of binding of 1 .mu.M glycine-stimulated [.sup.3 H]-MK-801 in rat or guinea pig brain membrane homogenates. The morepotent the glycine antagonist, the less [.sup.3 H]-MK-801 can bind since the [.sup.3 H]-MK801 binding site (PCP receptor) is accessible only upon opening of the ion channel by glutamate and glycine (Fletcher, E. L., et al., in Glycine Neurotransmission,Otterson, P., et al. (eds.), John Wiley and Sons (1990); Johnson, J. W., et al., Nature 325:529 (1987)).

1,2,3,4-Tetrahydroquinoline-2,3,4-trione-3-oxime 17a (Dahn, H. and Donzel, A., Helv. Chim. Acta 50:1911-1917 (1967)) was prepared bit reaction of 2,4-quinolinediol (Aldrich) 15a with NaNO.sub.2 /H.sub.2 SO.sub.4 (eq.8). .sup.1 H NMR shows that17a is a mixture of two stereoisomers 17aa and 17ab in a ratio of 0.3:0.7 (the specific isomers were not determined). All the other 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oximes prepared also are mixture of two isomers. Compound 17a was found tohave a K.sub.i value of 20 .mu.M with a potency of 1.3% of DCK. ##STR6##

5,7-Dichloro-2,4-quinolinediol 15b (the tautomer 5,7-Dichloro-4-hydroxy-1,2-dihydroquinolin-2-one is depicted in the following scheme) was prepared by reaction of 3,5-dichloroaniline with diethyl malonate followed by basic hydrolysis andcyclization in polyphosphoric acid (PPA) (Patel, G. H. and Mehta, C. M., J. Sci. Industr. Res. 19B:436-438 (1960)). 5,7-Dichloro-1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oxime 17b was prepared from 15b by reaction with NaNO.sub.2 /H.sub.2 SO.sub.4(eq. 9). 17b was found to be highly active as an antagonist for the glycine/NMDA receptor. It has a K.sub.i value of 0.02 .mu.M, which is about 1000 times more active than the unsubstituted compound 17a and is 1126% of DCK. Similar to 17a, 17b alsois a mixture of two stereoisomers in a ratio of 0.4:0.6. ##STR7##

A mixture of about 1:1 of 6,7-dichloro-2,4-quinolinediol 15c and 5,6-dichloro-2,4-quinolinediol 15d were obtained by reaction of 3,4-dichloroaniline with diethyl malonate under similar conditions as that of 15b. Nitrosation of the mixture of 15cand 15d gave a mixture of 17c and 17d (eq 10), which was separated by crystallization (95% ethanol). 6,7-Dichloro-1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oxime 17c was found to be highly active as an antagonist for the glycine/NMDA receptor. It hasa K.sub.i value of 0.05 .mu.M, which is 439% of DCK. 5,6-Dichloro-1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oxime 17d was active as an antagonist for the glycine/NMDA receptor. It has a K.sub.i value of 0.36 .mu.M, which is about 55% of DCK. 5,6,7-Triehloro-1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oxime was also prepared and found to exhibit a K.sub.i of 0.05 .mu.M, which is about 400% of DCK. ##STR8##

The acetyl derivative 18b was prepared by reacting 17b with acetic anhydride (eq 11). The reaction converts the ionizable NOH to an ester. It is possible that ester 18b might pass the blood-brain barrier easier than 17b. Ester 18b was found tobe active with a K.sub.i of 0.16 .mu.M, which is 142% of DCK. The ester also might hydrolyze under physiological conditions to produce the highly active 17b in vivo, thus 18b may be a prodrug. ##STR9##

1,2,3,4-Tetrahydroquinoline-2,3,4-trione-4-oxime 14a was prepared by nitrosation of 2,3-dihydroxyquinoline 11a. Compound 11a was prepared by ring expansion of isatin 10a with diazomethane followed by hydrolysis (eq 12). 1,2,3,4-Tetrahydroquinoline-2,3,4-trione-4-oxime 14a was found to be highly active with IC.sub.50 of 18.4 .mu.M, which is 14% of DCK. For all the known glycine/NMDA antagonists with no substituent in the benzene portion of the molecule, 14a is among themost active. ##STR10##

Thus, the present invention is directed to compounds having high binding to the glycine receptor and low binding to the kainate and AMPA sites. Particular compounds of the invention may have high antagonist potency at the kainate, AMPA andquisqualate receptors in addition to the glycine receptor. According to the present invention, those compounds having high binding to the glycine receptor exhibit a glycine binding affinity (K.sub.i) of about 100 .mu.M or less in a glycine bindingassay. Preferably, the compounds of the present invention exhibit a K.sub.i of 10 .mu.M or less. Most preferably, the compounds of the present invention exhibit a K.sub.i of 1 .mu.M or less. The compounds exhibit high binding to the kainate and AMPAsites if they exhibit a K.sub.i of about 10 .mu.M or less, especially, 1 .mu.M or less in a kainate or AMPA binding assay.

The glycine antagonist potency in vitro may be determined using a 1 .mu.M glycine-stimulated [.sup.3 H]-MK801 binding assay. This assay takes advantage of the fact that the binding of [.sup.3 H]-MK801 to the PCP receptor inside the pore of theNMDA channel is dependent on the presence of both glutamate and glycine. In the absence of glycine but in the presence of glutamate, [.sup.3 H]-MK801 cannot bind effectively to the PCP receptor, because the NMDA channel remains closed and access of[.sup.3 H]-MK801 to the PCP receptor inside the closed channel pore is severely restricted.

The assay is conducted using rat brain membrane homogenates which are enriched in NMDA receptors. The membranes are prepared as follows. Frozen rat brains (obtained from Pel-Freez, Rogers, Ark.) are homogenized in 15 volumes (w/v) of ice cold0.32M sucrose. The homogenate is spun at 1,000.times.g for ten minutes. The supernatant is collected and spun for 20 minutes at 44,000.times.g. The pellet is suspended in 15 volumes of water (relative to original brain weight). The homogenate isagain spun at 44,000.times.g for twenty minutes. The pellet is resuspended in 5 volumes of water and the suspension is freeze-thawed 2 times. After the final thaw cycle, the suspension is brought to 15 volumes with water and spun at 44,000.times.g fortwenty minutes. The pellet is resuspended in 5 volumes of ice-cold 10 mM HEPES, and is titrated to pH 7.4 with KOH containing 0.04% Triton X-100. Membranes are incubated with the Triton/HEPES buffer at 37.degree. C. for 15 minutes. The volume is thenbrought to 15 with ice-cold 10 mM HEPES, pH 7.4, and spun/washed three times with spins of 44,000.times.g between washes. The final pellet is suspended in three volumes of 50 mM HEPES, pH 7.4 and the protein concentration is determined with a standarddye-binding protein assay (Bio-Rad, Richmond, Calif.). The suspension is stored at -80.degree. C. until used. Only HPLC grade water is used for all buffers and suspensions/washings. The extensive washings are necessary to remove as much endogenousglycine from the membrane preparation as possible.

On the day of the assay, the previously prepared membranes are thawed and 5 mM Tris/HCl buffer, pH 7.4, is added to yield a final protein concentration of 0.156 mg/ml. For binding assays, 0.8 ml of membranes are pipetted into polypropylene tubesfollowed by 0.033 ml of 15.1 .mu.M 5,7-dichlorokynurenic acid (DCK), 0.033 ml of 30.3 .mu.M glycine in buffer (or buffer alone), 0.033 ml of 303 .mu.M glutamate in buffer (or for controls, 0.1 ml 1 mM PCP instead of DCK/gly/glu), 0.033 ml glycineantagonist in buffer (or buffer alone) and 0.1 ml buffer containing 200,000 cpm [.sup.3 H]-MK801. Nonspecific binding is defined as the difference in binding that occurs in the absence or presence of PCP (final concentration: 100 .mu.M). To determinethe effect of 1 .mu.M glycine on the binding of [.sup.3 H]-MK801, bound radioactivity in the presence of 10 .mu.M glutamate alone (final concentration) is subtracted from the bound radioactivity in the presence of both 10 .mu.M glutamate and 1 glycine(final concentration). A 500 nM concentration (final) of 5,7-dichlorokynurenic (DCK) acid is added to all assay tubes. This concentration of the glycine antagonist DCK "buffers" most of the residual endogenous glycine that is not removed by theextensive washing steps that are carried out during the membrane preparation procedure. The 500 nM DCK does not interfere with the stimulation of [.sup.3 H]-MK801 binding that is effected by the addition of 1 .mu.M exogenous glycine.

The assays are incubated for 120 minutes at room temperature after which time the membrane-bound radioactivity is isolated from the free radioactivity by vacuum filtration through Whatman glass fiber filters that had been pretreated with 0.3%polyethyleneimine. Filtration is accomplished using a Brandel 48 well cell harvester. Filtered membranes are washed three times with 3 ml each of ice cold buffer. Filters are transferred to scintillation vials and 5 ml of scintillation cocktail isadded. The vials are shaken overnight and the radioactivity is counted by liquid scintillation spectroscopy. The assays are done in triplicate and all experiments are conducted at least three times.

Inhibition dose response curves are constructed using increasing concentrations of glycine antagonists from 5 nM to 330 .mu.M. IC.sub.50 values are determined for compounds active in inhibiting 1 .mu.M glycine-stimulated [.sup.3 H]-MK801 bindingby computer-assisted plotting of the inhibition curves and interpolation. When compounds are found to inhibit glycine-stimulated [.sup.3 H]-MK801 binding, experiments are conducted to determine whether the inhibition of the glycine-stimulated [.sup.3H]-MK801 binding is indeed mediated at the glycine binding site of the NMDA receptor. In these experiments, a fixed concentration of antagonist sufficient to produce a >95% inhibition of the 1 .mu.M glycine-stimulated [.sup.3 H]-MK801 binding isincubated with the membranes without any additional glycine (above 1 .mu.M) and in the presence of increasing concentrations of additional glycine (2 .mu.M to 1 .mu.M). If the inhibition of [.sup.3 H]-MK801 binding by the drug in the presence of 1 .mu.Mglycine is fully reversed by adding increasing concentrations of glycine, then the inhibition of [.sup.3 H]-MK801 binding is mediated by the drug acting as an antagonist at the glycine binding site of the NMDA receptor.

After constructing inhibition dose response curves and determination of glycine reversibility, K.sub.i values for the glycine antagonists are calculated using the Cheng and Prusoff equation employing the experimentally determined IC.sub.50values, the known concentration of glycine in the assay (1 .mu.M) and the known affinity of glycine for the glycine binding site of the NMDA receptor (100 nM).

The same rat brain membrane homogenates used for the 1 .mu.M glycine-stimulated [.sup.3 H]-MK801 binding assay are used for the [.sup.3 H]-AMPA radioligand binding assay. On the day of the assay the frozen membranes (prepared as described above)are thawed and diluted with 30 mM Tris/HCl buffer containing 2.5 mM CaCl.sub.2 and 100 mM KSCN, pH 7.4, to yield a final membrane concentration of 1.25 mg/ml membrane protein. For the binding assay, 0.8 ml of membrane homogenate is added topolypropylene tubes followed by 0.033 ml drug and 0.067 ml buffer (or for controls by 0.1 ml buffer alone) and 0.1 ml buffer containing 200,000 cpm of [.sup.3 H]-AMPA. The assay is incubated for 30 minutes on ice. Bound radioactivity is separated fromfree radioactivity by filtration over Whatman glass fiber filters (pretreated with 0.3% polyethyleneimine) using a Brandel 48 well cell harvester.

Filtered membranes are washed three times with 3 ml each of ice cold buffer. The filters are transferred to scintillation vials and 5 ml of scintillation cocktail is added. The vials are shaken overnight and radioactivity is counted by liquidscintillation spectroscopy. Nonspecific binding is determined by the radioactivity that remains bound to the membranes in the presence of 10 mM glutamate. Inhibition dose response curves are constructed by adding increasing concentrations of drug from10 nM to 100 .mu.M.

The same membrane preparation as that used for the [.sup.3 H]-AMPA binding assay may be used for the [.sup.3 H]-Kainate radioligand binding assay. On the day of the assay the frozen rat brain membranes are thawed and 5 mM Tris/HCl buffer, pH7.4, is added to yield a final concentration of 0.5 mg/ml membrane protein. For the binding assay, 0.8 ml of membrane homogenate is added to polypropylene tubes followed by 0.033 ml drug and 0.067 ml buffer (or for controls by 0.1 ml buffer alone) and0.1 ml buffer containing 200,000 cpm of [.sup.3 H]-kainate. The assay is incubated for 2 hours on ice. Bound radioactivity is separated from free radioactivity by filtration over Whatman glass fiber filters (pretreated with 0.3% polyethyleneimine)using a Brandel 48 well cell harvester. Filtered membranes are washed three times with 3 ml each of ice cold buffer. The filters are transferred to scintillation vials and 5 ml of scintillation cocktail is added. The vials are shaken overnight andradioactivity is counted by liquid scintillation spectroscopy. Nonspecific binding is determined by the radioactivity that remains bound to the membranes in the presence 10 mM glutamate. Inhibition dose response curves are constructed by addingincreasing concentrations of drug from 250 nM to 330 .mu.M.

The anxiolytic activity of any particular compound of the present invention may be determined by use of any of the recognized animal models for anxiety. A preferred model is described by Jones, B. J. et at., Br. J. Pharmacol. 93:985-993(1988). This model involves administering the compound in question to mice which have a high basal level of anxiety. The test is based on the finding that such mice find it aversive when taken from a dark home environment in a dark testing room andplaced in an area which is painted white and brightly lit. The test box has two compartments, one white and brightly illuminated and one black and non-illuminated. The mouse has access to both compartments via an opening at floor level in the dividerbetween the two compartments. The mice are placed in the center of the brightly illuminated area. After locating the opening to the dark area, the mice are free to pass back and forth between the two compartments. Control mice tend to spend a largerproportion of time in the dark compartment. When given an anxiolytic agent, the mice spend more time exploring the more novel brightly lit compartment and exhibit a delayed latency to move to the dark compartment. Moreover, the mice treated with theanxiolytic agent exhibit more behavior in the white compartment, as measured by exploratory rearings and line crossings. Since the mice can habituate to the test situation, naive mice should always be used in the test. Five parameters may be measured:the latency to entry into the dark compartment, the time spent in each area, the number of transitions between compartments, the number of lines crossed in each compartment, and the number of rears in each compartment. The administration of thecompounds of the present invention is expected to result in the mice spending more time in the larger, brightly lit area of the test chamber.

In the light/dark exploration model, the anxiolytic activity of a putative agent can be identified by the increase of the numbers of line crossings and rears in the light compartment at the expense of the numbers of line crossings and rears inthe dark compartment, in comparison with control mice.

A second preferred animal model is the rat social interaction test described by Jones, B. J. et al., supra, wherein the time that two mice spend in social interaction is quantified. The anxiolytic activity of a putative agent can be identifiedby the increase in the time that pairs of male rats spend in active social interaction (90% of the behaviors are investigatory in nature). Both the familiarity and the light level of the test arena may be manipulated. Undrugged rats show the highestlevel of social interaction when the test arena is familiar and is lit by low light. Social interaction declines if the arena is unfamiliar to the rats or is lit by bright light. Anxiolytic agents prevent this decline. The overall level of motoractivity may also be measured to allow detection of drug effects specific to social behaviors.

The efficacy of the glycine and excitatory amino acid antagonists to inhibit glutamate neurotoxicity in rat brain cortex neuron cell culture system may be determined as follows. An excitotoxicity model modified after that developed by Choi(Choi, D. W., J. Neuroscience 7:357 (1987)) may be used to test anti-excitotoxic efficacy of the glycine and excitatory amino acid antagonists. Fetuses from rat embryonic day 19 are removed from tired mated pregnant rats. The brains are removed fromthe fetuses and the cerebral cortex is dissected. Cells from the dissected cortex are dissociated by a combination of mechanical agitation and enzymatic digestion according to the method of Landon and Robbins (Methods in Enzymology 124:412 (1986)). Thedissociated cells are passed through an 80 micron nitex screen and the viability of the cells are assessed by Trypan Blue. The cells are plated on poly-D-lysine coated plates and incubated at 37.degree. C. in an atmosphere containing 91% O.sub.2 /9%CO.sub.2. Six days later, fluoro-d-uracil is added for two days to suppress non-neural cell growth. At culture day 12, the primary neuron cultures are exposed to 100 .mu.M glutamate for 5 minutes with or without increasing doses of glycine andexcitatory amino acid antagonist or other drugs. After 5 minutes the cultures are washed and incubated for 24 hours at 37.degree. C. Neuronal cell damage is quantitated by measuring lactate dehydrogenase (LDH) activity that is released into the culturemedium. The LDH activity is measured according to the method of Decker et al. (Decker et al., J. Immunol. Methods 15:16 (1988)).

The anticonvulsant activity of the glycine and excitatory amino acid antagonists may be assessed in the audiogenic seizure model in DBA-2 mice as follows. DBA-2 mice may be obtained from Jackson Laboratories, Bar Harbor, Me. These mice at anage of <27 days develop a tonic seizure within 5-10 seconds and die when they are exposed to a sound of 14 kHz (sinus wave) at 110 dB (Lonsdale, D., Dev. Pharmacol. Ther. 4:28 (1982)). Seizure protection is defined when animals injected with drug30 minutes prior to sound exposure do not develop a seizure and do not die during a 1 minute exposure to the sound. 21 day old DBA-2 mice are used for all experiments. Compounds are given intraperitoneally in either saline, DMSO orpolyethyleneglycol-400. Appropriate solvent controls are included in each experiment. Dose response curves are constructed by giving increasing doses of drug from 1 mg/kg to 100 mg/kg. Each dose group (or solvent control) consists of at least sixanimals;

The anticonvulsant efficacy of the glycine receptor antagonists may be assessed in the pentylenetetrazol (PTZ)-induced seizure test as follows. Swiss/Webster mice, when injected with 50 mg/kg PTZ (i.p.) develop a minimal clonic seizure Ofapproximately 5 seconds in length within 5-15 minutes after drug injection. Anticonvulsant efficacy of a glycine/excitatory amino acid antagonist (or other) drug is defined as the absence of a seizure when a drug is given 30 minutes prior to PTZapplication and a seizure does not develop for up to 45 minutes following PTZ administration. Glycine/excitatory amino acid antagonist or other drugs are given intraperitoneally in either saline, DMSO or polyethyleneglycol-400. Appropriate solventcontrols are included in each experiment. Dose response curves are constructed by giving increasing doses of drug from 1 mg/kg to 100 mg/kg. Each dose group (or solvent control) consists of at least six animals.

The efficacy of glycine/excitatory amino acid antagonists to protect mice from NMDA-induced death may be assessed as follows. When mice are injected with 200 mg/kg N-methyl-D-asparmte (NMDA) i.p., the animals will develop seizures followed bydeath within 5-10 minutes. Glycine/excitatory amino acid antagonists are tested for their ability to prevent NMDA-induced death by giving the drugs i.p. 30 minutes prior to the NMDA application. Glycine/excitatory amino acid antagonist or other drugsare given intraperitoneally in either saline, DMSO or polyethyleneglycol-400. Appropriate solvent controls are included in each experiment. Dose response curves are constructed by giving increasing doses of drug from 1 mg/kg to 100 mg/kg. Each dosegroup (or solvent control) consists of at least six animals.

A series of different evaluations may be conducted on doses of the glycine/excitatory amino acid antagonists of the invention to determine the biological activity of the compounds both in normal gerbils and in animals exposed to 5 minutes ofbilateral carotid occlusion. See Scheme I. ##STR11##

These studies are conducted in animals who are conscious and have no other pharmacological agents administered to them. Gerbils are preinstrumented 48-hours prior to ischemia to allow for the complete elimination of the pentobarbital anestheticwhich is employed. When tested with drugs, animals are given IP injections of the glycine/excitatory amino acid antagonist or vehicle. In the case of multiple injections, animals are given IP injections 2 hours apart and the final injection is given 30minutes prior to the ischemic period or in the case of post treatment, the animals are given injections at 30 minutes, 2 hours, 4 hours and 6 hours post-ischemic reperfusion.

In order to assess the direct pharmacological activity or potential activity of the glycine/excitatory amino acid antagonists, naive gerbils are injected with either saline or differing doses of the antagonist. The behavioral changes areassessed using a photobeam locomotor activity chamber which is a two foot circular diameter arena with photobeam detection. Animals are individually placed in the 2 foot diameter chambers. The chambers are housed in a cabinet which is closed and noiseis abated using both a background white noise generator and a fan. Animals are placed in these chambers in the case of the initial pharmacological evaluation for a period of 6 hours and the total activity during each successive hour is accumulated usingthe computer control systems.

Saline results in an initial high rate of activity, with the control animals showing a first hour activity lextel of about 1600 counts. This level of control activity is typical for the gerbil under these experimental conditions. As the sessionprogressed, animals decrease their exploratory activity and at the terminal period the activity declines to about 250 counts per hour. It is expected that the glycine/excitatory amino acid antagonists of the present invention will have no significanteffect on either the initial exploratory rate or the terminal rate of exploration.

In a next phase of the evaluation of the glycine/excitatory amino acid antagonists, gerbils are pretreated with varying doses of the antagonists and then exposed to a five minute period of bilateral carotid occlusion. Following the initiation ofreperfusion, animals are placed into the circular locomotor activity testing apparatus and the activity at the beginning of the first hour following reperfusion is monitored for the subsequent four hours.

Control animals not exposed to ischemia and given injections of saline prior to being placed in the locomotor activity chamber show a characteristic pattern of activity which in the first hour of locomotor activity is substantially higher thanduring all other hours and progressively declined over the four hours to a very low value. In contrast to the progressive decline in activity over the four hour testing period, control animals that are exposed to five minutes of cortical ischemiademonstrate a completely different pattern of locomotor activity. During the first hour there is a significant decline in activity which is followed by a progressive increase in which the activity during the fourth hour is ten-fold higher than thatdemonstrated by animals not exposed to carotid occlusion. These results are typical and are a reliable result of the alterations caused by five minutes of bilateral carotid occlusion in the gerbil.

Separate groups of gerbils are pretreated with the glycine/excitatory amino acid antagonists of the invention 30 minutes before the onset of carotid occlusion and then placed into the locomotor activity following one hour of reperfusion. It isexpected that pretreatment of the gerbils with the glycine/excitatory amino acid antagonists of the invention will prevent both the post-ischemic decrease and increase in activity. Post-ischemic decreases in activity are expected to be near zero duringthe first hour following reperfusion. Pretreatment with the glycine/excitatory amino acid antagonists of the invention is expected to reduce or prevent this early depression of behavior. In addition, the glycine/excitatory amino acid antagonists of theinvention are expected to prevent the post-ischemic stimulation of behavior.

Subsequent to completion of the single dose pretreatment evaluations, gerbils are also evaluated with multiple injections of the glycine/excitatory amino acid antagonists of the invention. Doses are administered I.P. at 6 hours, 4 hours, 2hours and 30 minutes prior to the onset of 5 minutes of ischemia.

At 24 hours all animals are evaluated for differences in patrolling behavior using an 8-arm radial maze. In this procedure, animals are placed into the center start chamber of the maze, the barrier removed and the amount of time and the numberof times the animal makes an error recorded prior to completion of exploration in all 8 arms of the maze. An error is defined as the revisiting of an arm by entering to the extent of the entire body without including tail by the animal. If the animalperseveres or fails to leave the arm for longer than five minutes, the session is terminated. In the control population of the animals, the number of errors and exploration of the maze with no prior experience (naive) is approximately 6 errors. This isan average value for an N of 28 gerbils. Following 5 minutes of bilateral carotid occlusion and testing at 24 hours, gerbils make an average number of errors of 21. When animals are pretreated with the glycine/excitatory amino acid antagonists of theinvention, there is expected to be a significant reduction in the number of errors made. There is also expected to be a significant sparing of the behavioral changes that are induced in the radial arm maze performance.

It is also expected that post treatment the glycine/excitatory amino acid antagonists of the invention will reduce the short term memory impairment 24 hours post ischemic/reperfusion.

The effects of 5 minutes of bilateral carotid occlusion on neuronal cell death in the dorsal hippocampus may be evaluated in animals 7 days after ischemia reperfusion injury. Previous studies have demonstrated that neuronal degeneration beginsto occur around 3 days following cerebral ischemia. By 7 days those neurons which have been affected and will undergo cytolysis and have either completed degeneration or are readily apparent as dark nuclei and displaced nuclei with eosinophiliccytoplasm with pycnotic nuclei. The lesion with 5 minutes of ischemia is essentially restricted within the hippocampus to the CA1 region of the dorsal hippocampus. The intermedial lateral zone of the horn is unaffected and the dentate gyms and/or inCA3 do not show pathology. Gerbils are anesthetized on day 7 following ischemia with 60 mg/kg of pentobarbital. Brains are perfused transcardiac with ice-cold saline followed by buffered paraformaldehyde (10%). Brains are removed, imbedded andsections made. Sections are stained with hematoxylin-eosin and neuronal cell counts are determined in terms of number of neuronal nuclei/100 micrometers. Normal control animals (not exposed to ischemia reperfusion injury) will not demonstrate anysignificant change in normal density nuclei within this region. Exposure to five minutes of bilateral carotid occlusion results in a significant reduction in the number of nuclei present in the CA1 region. In general, this lesion results in a patchynecrosis instead of a confluent necrosis which is seen if 10 minutes of ischemia is employed. Pretreatment with the glycine receptor antagonists of the invention are expected to produce a significant protection of hippocampal neuronal degeneration.

It is known that NMDA receptors are critically involved in the development of persistent pain following nerve and tissue injury. Tissue injury such as that caused by injecting a small amount of formalin subcutaneously into the hindpaw of a testanimal has been shown to produce an immediate increase of glutamate and aspartate in the spinal cord (Skilling, S. R., .et al., J. Neurosci. 10:1309-1318 (1990)). Administration of NMDA receptor blockers reduces the response of spinal cord dorsal hornneurons following formalin injection (Dickenson and Aydar, Neuroscience Lett. 121:263-266 (1991); Haley, J. E., et al., Brain Res. 518:218-226 (1990)). These dorsal horn neurons are critical in carrying the pain signal from the spinal cord to thebrain and a reduced response of these neurons is indicative of a reduction in pain perceived by the test animal to which pain has been inflicted by subcutaneous formalin injection.

Because of the observation that NMDA receptor antagonists can block dorsal horn neuron response induced by subcutaneous formalin injection, NMDA receptor antagonists have potential for the treatment of chronic pain such as pain which is caused bysurgery or by amputation (phantom pain) or by infliction of other wounds (wound pain). However, the use of conventional NMDA antagonists such as MK801 or CGS 19755, in preventing or treating chronic pain, is severely limited by the adverse PCP-likebehavioral side effects that are caused by these drugs. It is expected that the glycine receptor antagonists that are the subject of this invention will be highly effective in preventing chronic pain in mice induced by injecting formalin subcutaneouslyinto the hindpaw of the animals. Because the glycine/excitatory amino acid antagonists of this invention are expected to be free of PCP-like side effects, these drugs are highly useful in preventing or treating chronic pain without causing PCP-likeadverse behavioral side effects.

The effects of the glycine receptor antagonists of the present invention on chronic pain may be evaluated as follows. Male Swiss/Webster mice weighing 25-35 grams are housed five to a cage with free access to food and water and are maintained ona 12 hour light cycle (light onset at 0800h). The glycine receptor antagonist is dissolved in DMSO at a concentration of 1-40 and 5-40 mg/ml, respectively. DMSO is used as vehicle control. All drugs are injected intraperitoneally (1 .mu.l/g). Theformalin test is performed as described (Dubuisson and Dennis, Pain 4:H161-174 (1977)). Mice are observed in a plexiglass cylinder, 25 cm in diameter and 30 cm in height. The plantar surface of one hindpaw is injected subcutaneously with 20 .mu.l of 5%formalin. The degree of pain is determined by measuring the amount of time the animal spends licking the formalin-injected paw during the following time intervals: 0-5' (early phase); 5'-10', 10'-15'and 15'-50'(late phase). To test whether theglycine/excitatory amino acid antagonists prevent chronic pain in the test animals, vehicle (DMSO) or drugs dissolved in vehicle at doses of 1 mg/kg to 40mg/kg are injected intraperitoneally 30 minutes prior to the formalin injection. For each dose ofdrug or vehicle control at least six animals are used.

Compared to vehicle control, it is expected that the intraperitoneal injection of the glycine receptor antagonists 30 minutes prior to formalin injection into the hindpaw will significantly inhibit formalin-induced chronic pain in adose-dependent manner as determined by the reduction of the time spent licking by the mouse of the formalin injected hindpaw caused by increasing doses of glycine/excitatory amino acid antagonist.

Compositions within the scope of this invention include all compositions wherein the compounds of the present invention are contained in an amount which is effective to achieve its intended purpose. While individual needs vary, determination ofoptimal ranges of effective amounts of each component is with the skill of the art. Typically, the compounds may be administered to mammals, e.g., humans, orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceutically acceptablesalt thereof, per day of the body weight of the mammal being treated for anxiety disorders, e.g., generalized anxiety disorder, phobic disorders, obsessional compulsive disorder, panic disorder, and post traumatic stress disorders. Preferably, about0.01 to about 10 mg/kg is orally administered to treat or prevent such disorders. For intramuscular injection, the dose is generally about one-half of the oral dose. For example, for treatment or prevention of anxiety, a suitable intramuscular dosewould be about 0.0025 to about 15 mg/kg, and most preferably, from about 0.01 to about 10 mg/kg.

In the method of treatment or prevention of neuronal loss in ischemia, brain and spinal cord trauma, hypoxia, hypoglycemia, and surgery, as well as for the treatment of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease andDown's Syndrome, or in a method of treating a disease in which the pathophysiology of the disorder involves hyperactivity of the excitatory amino acids or NMDA receptor-ion channel related neurotoxicity or psychosis, the pharmaceutical compositions ofthe invention may comprise the compounds of the present invention at a unit dose level of about 0.01 to about 50 mg/kg of body weight, or an equivalent amount of the pharmaceutically acceptable salt thereof, on a regimen of 1-4 times per day. When usedto treat chronic pain or to induce anesthesia, the compounds of the invention may be administered at a unit dosage level of from about 0.01 to about 50 mg/kg of body weight, or an equivalent amount of the pharmaceutically acceptable salt thereof, on aregimen of 1-4 times per day. Of course, it is understood that the exact treatment level will depend upon the case history of the animal, e.g., human being, that is treated. The precise treatment level can be determined by one of ordinary skill in theart without undue experimentation.

The unit oral dose may comprise from about 0.01. to about 50 mg, preferably about 0.1 to about 10 mg of the compound. The unit dose may be administered one or more times daily as one or more tablets each containing from about 0.1 to about 10,conveniently about 0.25 to 50 mg of the compound or its solvates.

In addition to administering the compound as a raw chemical, the compounds of the invention may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliarieswhich facilitate processing of the compounds into preparations which can be used pharmaceutically. Preferably, the preparations, particularly those preparations which can be administered orally and which can be used for the preferred type ofadministration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent,preferably from about 0.25 to 75 percent of active compound(s), together with the excipient.

Also included within the scope of the present invention are the non-toxic pharmaceutically acceptable salts of the compounds of the present invention. Basic salts are formed by mixing a solution of the particular1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 or 4-oxime the present invention with a solution of a pharmaceutically acceptable non-toxic base such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium bicarbonate, sodium carbonate, Tris andthe like.

The pharmaceutical compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended tobe so limited.

The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,transdermal, buccal or ocular routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequencyof treatment, and the nature of the effect desired.

When the compositions of the invention are administered ocularly, one may achieve either local or systemic administration. For example, the compositions of the present invention may be administered in the form of eye drops which aresubstantially isotonic with tear fluid to achieve systemic administration. Preferably, such compositions will also comprise a permeation-enhancing agent which aids the systemic absorption of the compounds of the present invention. See, U.S. Pat. No.5,182,258. Alternatively, the compositions of the invention may be administered ocularly to treat or prevent optic nerve degeneration. In this embodiment, the compounds of the present invention are administered in the form of eye drops, as disclosedabove, or may be injected into the vicinity of the optic nerve. In the alternative, thin ocular implants may be employed which slowly release the compounds of the present invention.

In addition to the pharmacologically active compounds, the new pharmaceutical preparations may contain suitable pharmaceutically acceptable carries comprising excipients and auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Preferably, the preparations, particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, andalso preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, are present at a concentration of from about 0.01 to 99 percent, together with the excipient.

The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceuticalpreparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtaintablets or dragee cores.

Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as wellas binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired,disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulatingagents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastricjuices. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solventmixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs or pigments may be added to the tabletsor dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.

Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer'such as glycerol or sorbitol. The push-fit capsules can contain the activecompounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, stabilizers may be ridded.

Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active compounds with a suppository base. Suitable suppository bases are, for example,natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquidtriglycerides, polyethylene glycols, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts and alkaline solutions. In addition, suspensions of the active compounds as appropriateoily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compoundsare soluble in PEG-400). Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also containstabilizers.

The characterization of glycine binding sites in vitro has been difficult because of the lack of selective drug ligands. Thus, the glycine ligands of the present invention may be used to characterize the glycine binding site. Particularlypreferred substituted and unsubstituted 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 or 4-oximes which may be used for this purpose are isotopically radiolabelled derivatives, e.g., where one or more of the atoms are replaced with .sup.3 H, .sup.11 C,.sup.14 C, .sup.15 N, or .sup.18 F.

The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapyand which are obvious to those skilled in the art are within the spirit and scope of the invention.

A solution of 11.6 g (71.6 mmol) of 3,5-dichloroaniline and 26.3 g (164 mmol)of diethyl malonate was heated at 180.degree. C. for 6 h and the ethanol produced was collected (.about.3.5 mL). The solution was cooled to room temperature andprecipitate was observed. The mixture was filtered and washed with methanol (40 mL). The filtrate was mixed with 26 g of Na.sub.2 CO.sub.3 and 200 mL of water and refluxed for 1 h. The solution was acidified with aqueous 2N HCL in an ice-bath to pH=1. The mixture was filtered and washed with water, and dried to leave a white solid (13.9 g). The solid was mixed with 150 mL of polyphosphoric acid and heated at 140.degree. C. for 3 h. The solution was cooled to room temperature and diluted with 200 mLof aqueous 1N HCl. The mixture was stirred for 4 h, then neutralized with aqueous 20% NaOH to pH=4.It was filtered and washed with water, and dried to leave 12.3 g (74%) of 15b as white solid, mp 360.degree.-361.degree. C. (decomposed). .sup.1 H NMR(CDCl.sub.3 +DMSO-d.sub.6), 5.447 (s, 1), 6.620 (d, 1, J=1.84), 6.804 (d, 1, J=1.86), 10.915 (sb, 1). MS, 229 (100, M.sup.+), 187 (80), 160 (30), 124 (20). High resolution MS, Calcd for C.sub.9 H.sub.5.sup.35 Cl.sub.2 NO.sub.2 228.9694, found 228.9709.

A solution of 9.75 g (60.2 mmol) of 3,4-dichloroaniline and 21.1 g (132 mmol) of diethyl malonate was heated at 180.degree. C. under N.sub.2 for 30 h. It was cooled to room temperature, filtered and washed with 10 mL of methanol. The filtratewas mixed with 15 g of Na.sub.2 CO.sub.3, 30 mL of water and 10 mL of methanol and refluxed for 1 h. The mixture was acidified with aqueous 6N HCl in an ice-bath to pH=1. The precipitate was filtered and washed with water, and dried to leave a whitesolid (9.4 g). .sup.1 H NMR (CDCl.sub.3 DMSO-d.sub.6), 3.099 (s, 2), 7.028 (d, 1, J=8.75), 7.128 (dd, 1, J=2.26, 8.70), 7.619 (d, 1, J=2.23), 9.800 (s, 1). The solid was mixed with 100 mL of polyphosphoric acid and heated at 140.degree. C. for 3 h.The solution was cooled to room temperature and diluted with 100 mL of aqueous 1N HCl and 300 mL of water in an ice-bath. The mixture was then neutralized in the ice-bath with aqueous 20% NaOH to pH=4. The mixture was filtered and washed with water,and dried to leave 8.2 g (95%) of pale-yellow solid as a mixture of 15c and 15d. .sup.1 H NMR (CDCl.sub.3 +DMSO-d.sub.6), 15c: 5.513 (s, 1), 7.050 (s, 1), 7.510 (s, 1), 10.861 (sb, 1); 15d: 5.597 (s, 1), 6.846 (d, 1, J=9.0), 7.066 (d, 1, J=9.0), 10.9(sb, 1); 15c:15d=1:1 A portion (800 mg) of the mixture was dissolved in 15 mL of aqueous 1N NaOH and filtered. The filtrate was acidified with aqueous 2N HCl to pH=8.2 to give a solid precipitate. It was filtered, washed with water and dried to leave awhite solid. .sup.1 H NMR shows 15c:15d=1:1. The aqueous solution was acidified to pH=8.0 to give more precipitate. It was filtered, washed with water and dried to leave a white solid. .sup.1 H NMR shows 15c:15d=1:1. The aqueous solution wasacidified to pH=4.0 to give more precipitate. It was filtered, washed with water and dried to leave a white solid. .sup.1 H NMR shows 15c:15d=1:0.7.

To a solution of 201 mg (0.776 mmole) of 17b in 2 mL of DMSO was added 0.4 mL of acetic anhydride and the solution was stirred overnight. Solid precipitate was observed after about 1 hr. To the mixture was added 10 mL of ice-water and themixture was stirred until all of the ice melted. The mixture was filtered and washed with water, and dried to leave 228 mg (97%) of 18b as a yellow solid, mp 194.degree.-195.degree. C. .sup.1 H NMR (DMSO-d.sub.6), 2.292 (s, 3), 7.095 (m, 1), 7.34 (m,1), 11.39 (s, 0.4), 11.51 (s, 0.6).

Example 5

Preparation of 1,2,3,4- Tetrahydroquinoline-2,3,4-trione4-oxime (14a)

2,3-quinolinediol (11a)

To a mixture of 1.05 g (7.1 mmol) of isatin (Aldrich) in 15 mL of ether kept in an ice-bath was added in one portion a solution of diazomethane in ether (prepared from 4.4 g of Diazald, containing approximately 13 mmol of diazomethane). Themixture was stirred in an ice-bath for 3 h, then at 25.degree. C. overnight. It was filtered and dried to leave a white solid 0.648 g (52%), mp 184.degree.-185.degree. C. .sup.1 H NMR, 3.977 (s, 3), 7.014 (s, 1), 7.21 (m, 1), 7.39 (m, 2), 7.509 (d, 1,J=7.8), 11.429 (s, 1). A solution of 301 mg of the above white solid in 1 mL of acetic acid and 1 mL of 48% HBr was refluxed for 2 days. It was cooled to room temperature to give a solid precipitate which was diluted with 2 mL of water, filtered andwashed with water, and dried to leave an almost colorless solid. The solid was dissolved in 2 mL of 2N NaOH to form a clear solution. It was acidified by the addition dropwise of 1N HCl to pH=6. The white precipitate was filtered and washed withwater, and dried to leave 250 mg (90%) of 11a as a white solid, mp 261.degree.-262.degree. C. .sup.1 H NMR (CDCl.sub.3 +DMSO-d.sub.6), 6.618 (s, 1), 6.698 (t, 1, J=7.5), 6.809-6.901 (m, 2), 6.985 (d, 1, J=7.8), 8.061 (s, 1), 11.448 (s, 1).

Preparation of 5,6,7-trichloro-1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oxime

3,4,5-Trichloroaniline monomalonamide

To a solution of 3,4,5-trichloroaniline (393 mg, 2.0 mmol) and triethylamine (680 mg. 6.7 mmol) in 30 mL of anhydrous ether was added a solution of methyl malonyl chloride. (920 mg. 7.4 mmol) in 10 mL of anhydrous ether dropwise with stirringat 25.degree. C. under N.sub.2. After this addition, the resulting mixture was stirred at 25.degree. C. for 16 h. then 20 mL of 0.5N aq. HCl was added and stirred for 0.5 h.

5,7-Dichloro-1,2,3,4-tetrahydroquinoline-2,3,4-trione-3-oxime was evaluated for anticonvulsant activity after i.p. administration in the mouse maximum electroshock seizure model. Mice, 30-35 g (male) were injected i.p. with 70 mg/kg of thedrug dissolved in DMSO (1 ul/g). Ten mice each were used in the drug and control groups. The control group was injected with an equal volume of DMSO. Seizures were elicited by stimulation with corneal electrodes. The stimulation parameters foreliciting the seizures were as follows: 50 mA, 60 pulses/second square wave, 0.8 ms pulse width, 0.2 sec length of stimulus. Under these conditions, 100% of control animals developed tonic hindlimb extension immediately after the corneal electricalstimulation. A Hugo Baslie electroshock seizure stimulator was used for these experiments. The seizure stimulus was applied 1 hour after injection of the drug. Under these conditions, the drug prevented seizures from occurring in 8 out of 10 animalsinjected with drug. No seizure protection was seen in the control (DMSO-only) group.

Example 8

In Vitro Binding Assay

Table 1 shows the results of binding assays at the glycine binding site for a number of 1,2,3,4-tetrahydroquinoline-2,3,4-trione-3 and 4-oximes.

Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scopeof the invention or any embodiment thereof. All patents and publications cited herein are fully incorporated by reference herein in their entirety.